Arie J. Beld

Stereospecific binding as a tool in attempts to localize and isolate muscarinic receptors: Part II. Binding of (+_)-benzetimide, (−)-benetimide and atropine to a fraction from bovine tracheal smooth muscle and to bovine caudate nucleus

The concentration-dependent binding of (+)-benzetimide-H3 and (−)-benzetimide-H3, the active and inactive enantiomers, respectively of the anticholinergic agent, (±)-benzetimide, and of atropine-3 to a fraction from bovine tracheal smooth muscle was studied by equilibrium dialysis. Analysis of the binding curves revealed the presence of a saturable high affinity binding site for (+)-benzetimide and atropine but not for (−)-benzetimide. The stereospecificity of the binding and the results of competition experiments with cholinergic and non-cholinergic compounds provided evidence, that the high affinity binding sites for (+)-benzetimide and atropine are identical with the muscarinic receptor. Attempts to solubilize the binding sites with detergents, commonly used in this type of work, resulted in complete loss of stereospecificity. Digitonin, a plant glycoside with mild detergent properties, released components to which (+)-benzetimide-H3 and (−)-benzetimide-H3 were still bound differently, strongly suggesting the presence of solubilized muscarinic receptors. The stereoselective binding sites for anticholinergic agents, as observed in the caudate nucleus, could also be solubilized with digitonin but only after hexane extraction of lyophilized homogenates.

The Pharmacon-Receptor-Effector Concept

The biological effect of chemical agents can only be the result of an interaction of the molecules of the active agent and particular molecules or molecular complexes present in the biological object. The same holds true for endogenous bioactive agents such as hormones, neurotransmitters, etc. Often these messengers have their molecular sites of action, referred to as their specific receptors, located on the outer cell membrane (Lefkowitz et al., 1976). They then transmit information from remote or nearby cells to the surface of the target cells where they trigger these cells, thus inducing certain changes in cell function. The interaction between the bioactive agent and its specific receptors initiates the transduction of information received from outside to the cell interior. An example is the generation of intracellular mediators such as cyclic AMP. Similarly, the hormone-receptor complex, in the case of the interaction of steroid hormones and their specific proteins in the cytoplasm, is involved in the transduction of information to the cell nucleus. The presence of specific receptor for the bioactive agents either on the outer cell membrane or in the cytoplasm of only particular tissue cells accounts for a selectivity in action on these tissues.

Autoradiographic visualization of muscarinic receptors in pulmonary nerves and ganglia

We investigated autoradiographically the distribution of muscarinic receptors in bovine airways using (-)-[3H]quinuclidinyl benzilate as radioligand. The autoradiographs demonstrated the presence of muscarinic receptors in smooth muscle as well as neuronal muscarinic receptors in pulmonary nerves and ganglia. It is reasonable to believe that the neuronal muscarinic receptors participate in the regulation of neurotransmitter release at the peripheral nerve terminals innervating the bronchial smooth muscle.

Quantitative correlations between chemical structure and affinity for acetylcholine receptors

The affinity constants (log K, pA2) of 128 quaternary ammonium compounds belonging to several different series have been correlated linearly with the hydrophobicity (piR) constant, the dipole moment (muR), and the number of hydroxyl groups (nOH) of the side chain; the dependence on the hydrophobicity constant of the quaternary ammonium head (pi-N identical to) is shown to be parabolic. A correlation coefficient of 0.96 is obtained for all the compounds using only 4 independent variables (6 terms). Based on the quantitative correlation obtained, intermolecular forces involved in the drug-receptor interaction are discussed. Further molecular modifications to enhance the affinity to cholinergic receptors are suggested.

Muscarinic receptors in rat nasal mucosa are predominantly of the low affinity agonist type.

Specific [3H]l-quinuclidinyl benzilate binding to rat nasal mucosa homogenates occurs to a homogeneous class of binding sites with Kd = 60 +/- 2 10(-12) M and Bmax = 8.1 +/- 2 pmol/g tissue. Binding is stereoselectively inhibited by benzetimide enantiomers. Pirenzepine inhibits [3H]l-quinuclidinyl benzilate binding with low affinity (Ki = 5.0 10(-7) M), classifying the binding sites as muscarinic M2-receptors. Methylfurtrethonium and methacholine inhibit [3H]l-quinuclidinyl benzilate binding following an almost sigmoid curve at high concentrations pointing to the presence of mainly low affinity agonist binding sites.

Beta adrenoceptor binding and induced relaxation in airway smooth muscle from patients with chronic airflow obstruction.

Beta adrenoceptor function in central airway smooth muscle of patients with chronic airflow obstruction was investigated by radioligand binding studies and isoprenaline relaxation experiments. Receptor characteristics were determined in tracheal smooth muscle preparations obtained at necropsy from 12 patients and in bronchial tissue obtained at thoracototomy from 21 patients with chronic airflow obstruction. Receptor characteristics were compared with those obtained in airway tissue preparations from 65 control subjects without chronic airflow obstruction. The number of beta adrenoceptors, their binding affinity for the radioligand [125I]-(-)-cyanopindolol, and the tissue binding characteristics of isoprenaline were similar in tissue from patients with chronic airflow obstruction and from control subjects. Isoprenaline induced relaxation of tracheal smooth muscle without precontraction by methacholine showed slightly (though not significantly) less sensitivity to isoprenaline in patients with chronic airflow obstruction than in control subjects (mean (SEM) pD2--the negative logarithm of the concentration producing 50% relaxation--6.32 (0.16) v 6.62 (0.15)). The same pattern of pD2 values was found in segmental bronchial strips without precontraction by methacholine (chronic airflow obstruction 6.55 (0.27), control 7.14 (0.12)). Isoprenaline relaxation in segmental bronchial strips when contracted maximally was significantly less in the patients with airflow obstruction than in the control subjects (pD2 value 5.99 (0.18) v 6.45 (0.07)). These results suggest that beta adrenoceptors in airway smooth muscle of patients with chronic airflow obstruction are not abnormal in number or in binding affinity but that there is less effective coupling between components of the relaxant system distal to the beta adrenoceptor. The possibility that the reduced isoprenaline sensitivity is a consequence of previous bronchodilator treatment cannot be excluded.

Ligand binding to dopamine-receptors: Analysis & interpretation

Abstract Dopamine receptor interaction was studied by concentration dependent inhibition of 3 H-Spiperone binding to calf caudate nucleus homogenates with (+)-butaclamol, haloperidol, ergometrine, apomorphine and dopamine. The results were analyzed in terms of a one and a two site receptor model using computerized curve fitting procedures. Evidence is given in favour of a two site receptor model on basis of statistical criteria. The presence of two independent non-interconverting receptor sites for dopamine in calf caudate nucleus is consistent with binding data from others and with in vivo experiments.

Muscarinic receptor binding in central airway musculature in chronic airflow obstruction

The hypothesis of an increased muscarinic receptor sensitivity in airway musculature of patients with chronic airflow obstruction (CAO) has been investigated by in vitro radioligand binding studies. The receptor binding profiles were determined in membrane homogenates of tracheal and main bronchial smooth muscle, as well as in segmental bronchial tissue preparations. The number of receptors was measured by binding of the radioactive muscarinic antagonist [3H]-(-)-quinuclidinyl benzilate ([3H]-(-)-QNB). The affinity for agonists was investigated by studying the inhibition of [3H]-(-)-QNB binding by the full muscarinic agonist methylfurtrethonium. Binding characteristics were determined (1) in tracheal smooth muscle from 7 patients with chronic airflow obstruction (2) in main bronchial smooth muscle of 5 patients with CAO and (3) in segmental bronchial tissue of 10 patients with CAO. The receptor binding properties were compared with values obtained on airway tissue from 31 subjects without CAO. Muscarinic receptors in smooth muscle preparations of trachea and main bronchus were present in normal density, and showed normal [3H]-(-)-QNB binding affinity and methylfurtrethonium binding properties. In segmental bronchial tissue preparations which contain smooth muscle and glandular tissue, also normal receptor numbers and [3H]-(-)-QNB affinity values were found. Morphometric examination of these preparations revealed normal amounts of smooth muscle and submucosal glands. This suggests that the excessive mucus production of CAO is not accompanied by an increased muscarinic receptor density in submucosal glands. These observations suggest unaltered muscarinic receptor characteristics in central airway smooth muscle of patients with chronic airflow obstruction.

β-adrenoceptors in human tracheal smooth muscle: characteristics of binding and relaxation

Specific binding of [125I]-(-)-cyanopindolol to human tracheal smooth muscle membranes was saturable, stereo-selective and of high affinity (Kd = 5.3 +/- 0.9 pmol/l and RT = 78 +/- 7 fmol/g tissue). The beta 1-selective antagonists atenolol and LK 203-030 inhibited specific [125I]-(-)-cyanopindolol binding according to a one binding site model with low affinity in nearly all subjects, pointing to a homogeneous beta 2-adrenoceptor population. In one subject using LK 203-030 a small beta 1-adrenoceptor subpopulation could be demonstrated. The beta-mimetics isoprenaline, fenoterol, salbutamol and terbutaline recognized high and low affinity agonist binding sites. Isoprenalines pKH- and pKL-values for the high and low affinity sites were 8.0 +/- 0.2 and 5.9 +/- 0.3 respectively. In functional experiments isoprenaline relaxed tracheal smooth muscle strips having intrinsic tone with a pD2-value of 6.63 +/- 0.19.

Inhibition of [3H] Dexetimide binding by a homologous series of methylfurthrethonium analogues at the peripheral muscarinic receptor

The interaction of a homologous series of methylfurthrethonium analogues, in which there is a gradual transition from full agonists via partial agonists to antagonists, with the peripheral muscarinic receptor was investigated by concentration-dependent inhibition of [3H] Dexetimide binding. The antagonists give normal sigmoid inhibition curves, but those of agonists follow a much flatter course. The results can be explained by assuming the presence of two non-interconverting muscarinic binding sites for which agonists have different and antagonists have identical affinities. The affinity ratio for the two binding sites decreases from 61 for methylfurthrethonium to 1 for its isopropyl analogue and parallels the decrease in intrinsic activity.